PURIFICATION AND CHARACTERIZATION OF THE MAJOR NONSTRUCTURAL PROTEIN (NS-1) OF ALEUTIAN MINK DISEASE PARVOVIRUS

被引:40
作者
CHRISTENSEN, J
PEDERSEN, M
AASTED, B
ALEXANDERSEN, S
机构
[1] STATE VET INST VIRUS RES,DK-4771 KALVEHAVE,DENMARK
[2] ROYAL VET & AGR UNIV,DEPT PHARMACOL & PATHOBIOL,MOLEC PATHOBIOL LAB,DK-1870 FREDERIKSBERG C,DENMARK
[3] NATL VET LAB,DK-1790 COPENHAGEN V,DENMARK
关键词
D O I
10.1128/JVI.69.3.1802-1809.1995
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
We have previously described the expression of the major nonstructural protein (NS-1) of Aleutian mink disease parvovirus (ADV) in insect cells by using a baculovirus vector (J. Christensen, T. Storgaard, B. Bloch, S. Alexandersen, and B. Aasted, J. Virol. 67:229-238, 1993). To study its biochemical properties, ADV NS-I was expressed in Sf9 insect eells and purified to apparent homogeneity with a combination of nuclear extraction, Zn2+ ion chromatography, and immunoaffinity chromatography on monoclonal antibodies. The purified protein showed ATP binding and ATPase- and ATP- or dATP-dependent helicase activity requiring either Mg2+ or Mn2+ as a cofactor. The ATPase activity of NS-1 was efficiently stimulated by single-stranded DNA and, to a lesser extent, double-stranded DNA. We also describe the expression, purification, and characterization of a mutant NS-1 protein, in which a lysine in the putative nucleotide binding consensus sequence of the molecule was replaced with serine. The mutated NS-1 was expressed at 10-fold higher levels than wild-type NS-1, but it exhibited no ATP binding, ATPase, or helicase activity. The availability of large amounts of purified functional NS-1 protein will facilitate studies of the biochemistry of ADV replication and gene regulation leading to disease in mink.
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页码:1802 / 1809
页数:8
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